Effect of cross-linkers on the structure and function of pig-renal sodium-glucose cotransporters after papain treatment.
Kidney brush-border membranes contain two sodium-dependent glucose transporters, one with low and one with high affinity for phlorizin, the specific inhibitor of these transporters. Using Scatchard analysis of phlorizin binding and Western blotting with specific antibodies against these transporters, we demonstrate in this study that although both transporters were proteolysed by papain treatment, only the high-affinity phlorizin-binding sites were decreased. Papain treatment followed by cross-linking with homobifunctional disuccinimidyl tartarate restored only the structure of the low-affinity phlorizin-binding protein (approx. molecular mass 70 kDa) without modifying the phlorizin-binding sites. When disuccinimidyl tartarate was replaced with dithiobis(succinimidyl acetate), another homobifunctional cross-linker with a higher spacer arm, the low- and high-affinity sites were both restored, with reappearance of two phlorizin-binding proteins with approx. molecular masses of 70 and 120 kDa. We conclude that high-affinity phlorizin-binding sites depend on the presence of the heterodimeric 120 kDa protein.
Giudicelli
J
J
Laboratoire de Biochimie, Faculté de Médecine, Avenue de Valombrose, F-06107 Nice Cedex 2, France.
Bertrand
M F
MF
Bilski
S
S
Tran
T T
TT
Poiree
J C
JC
D000818:Animals; D001665:Binding Sites; D005947:Glucose / Q000378:metabolism*; D007421:Intestine, Small / Q000378:metabolism*; D007672:Kidney Cortex / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008871:Microvilli / Q000378:metabolism; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D010206:Papain / Q000378:metabolism*; D010695:Phlorhizin / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D051269:Sodium-Glucose Transporter 1; D013329:Structure-Activity Relationship; D013552:Swine
D008562:Membrane Glycoproteins; D009004:Monosaccharide Transport Proteins; D051269:Sodium-Glucose Transporter 1; D012964:Sodium; D010695:Phlorhizin; D010206:Papain; D005947:Glucose